phosphate solution in 900 mL deionized water. Adjust pH to
7.0 with HCl and add deionized water to a final volume of 1 L.
Sterilize by autoclaving for 20 min and store at room temperature. When preparing TAP agar plates, add 1.5% agar powder
prior to autoclaving of the medium.
5. TAP agar (1.5%) plates.
6. Gyratory shaker.
2.3 Transformation
1. TAP agar (1.5%) plate containing 30 μg/mL of hygromycin B.
2. Transformation buffer: TAP medium with 40 mM Sucrose.
Store the buffer at 4
C.
3. Recovery buffer: TAP medium with 40 mM Sucrose. Store at
room temperature.
4. Cell fix solution: Mix 178 μL of TAP medium and 20 μL of 10%
glutaraldehyde in 1.5 mL tube.
5. 2 mm gap electroporation cuvette (Nepa Gene Co., Ltd.):
Prechill the cuvette at 4
C.
6. Electroporator, NEPA21 (Nepa Gene Co., Ltd.).
7. Rotary shaker.
8. Hemocytometer.
9. 14 mL culture tube (sterile).
10. 300 mL flask (sterile).
11. 1.5 mL tube (sterile).
12. 15 mL conical tube (sterile).
13. 50 mL conical tube (sterile).
14. Swing-rotor centrifuge.
15. Chlamydomonas cell-walled wild-type strain (see Note 1).
3 Methods
3.1 Preparation
of aph7
00 DNA Cassette
1. Mix 10 μL of 5Â PrimeSTAR GXL buffer, 4 μL of dNTP
mixture (2.5 mM each), 1.5 μL of pHyg3-F1 primer
(10 μM), 1.5 μL of pHyg3-R1 primer (10 μM), 1 μL of
pHyg3 plasmid, 0.5 μL of PrimeSTAR GXL DNA Polymerase,
and 31.5 μL of distilled water in the PCR tube.
2. Amplify the aph7
00 DNA cassette by 35 cycles of denaturation
for 10 s at 98
C, annealing for 15 s at 60
C, and extension for
2 min at 68
C.
3. Check the PCR product (1,999 bp) by 1% agarose/TAE gel
electrophoresis.
4. Purify the PCR product by QIAquick PCR Purification Kit and
adjust the concentration to 25 ng/μL.
Transformation of Chlamydomonas reinhardtii
157